Homologous recombination contributes to the repair of acetaldehyde-induced DNA damage.

Acetaldehyde, a chemical that can cause DNA damage and contribute to cancer, is prevalently present in our environment, e.g. in alcohol, tobacco, and food. Although aldehyde potentially promotes crosslinking reactions among biological substances including DNA, RNA, and protein, it remains unclear what types of DNA damage are caused by acetaldehyde and how they are repaired. In this study, we explored mechanisms involved in the repair of acetaldehyde-induced DNA damage by examining the cellular sensitivity to acetaldehyde in the collection of human TK6 mutant deficient in each genome maintenance system. Among the mutants, mismatch repair mutants did not show hypersensitivity to acetaldehyde, while mutants deficient in base and nucleotide excision repair pathways or homologous recombination (HR) exhibited higher sensitivity to acetaldehyde than did wild-type cells. We found that acetaldehyde-induced RAD51 foci representing HR intermediates were prolonged in HR-deficient cells. These results indicate a pivotal role of HR in the repair of acetaldehyde-induced DNA damage. These results suggest that acetaldehyde causes complex DNA damages that require various types of repair pathways. Mutants deficient in the removal of protein adducts from DNA ends such as TDP1-/- and TDP2-/- cells exhibited hypersensitivity to acetaldehyde. Strikingly, the double mutant deficient in both TDP1 and RAD54 showed similar sensitivity to each single mutant. This epistatic relationship between TDP1-/- and RAD54-/- suggests that the protein-DNA adducts generated by acetaldehyde need to be removed for efficient repair by HR. Our study would help understand the molecular mechanism of the genotoxic and mutagenic effects of acetaldehyde.

Homology recognition without double-stranded DNA-strand separation in D-loop formation by RecA.

RecA protein and RecA/Rad51 orthologues are required for homologous recombination and DNA repair in all living creatures. RecA/Rad51 catalyzes formation of the D-loop, an obligatory recombination intermediate, through an ATP-dependent reaction consisting of two phases: homology recognition between double-stranded (ds)DNA and single-stranded (ss)DNA to form a hybrid-duplex core of 6-8 base pairs and subsequent hybrid-duplex/D-loop processing. How dsDNA recognizes homologous ssDNA is controversial. The aromatic residue at the tip of the ß-hairpin loop (L2) was shown to stabilize dsDNA-strand separation. We tested a model in which dsDNA strands were separated by the aromatic residue before homology recognition and found that the aromatic residue was not essential to homology recognition, but was required for D-loop processing. Contrary to the model, we found that the double helix was not unwound even a single turn during search for sequence homology, but rather was unwound only after the homologous sequence was recognized. These results suggest that dsDNA recognizes its homologous ssDNA before strand separation. The search for homologous sequence with homologous ssDNA without dsDNA-strand separation does not generate stress within the dsDNA; this would be an advantage for dsDNA to express homology-dependent functions in vivo and also in vitro.

Cell fusion upregulates PD-L1 expression for evasion from immunosurveillance.

MSCs (mesenchymal stem cells), responsible for tissue repair, rarely undergo cell fusion with somatic cells. Here, we show that ~5% of bladder cancer cells (UMUC-3) fuses with bone marrow-derived MSC (BM-MSC) in co-culture and maintains high tumorigenicity. In eleven fusion cell clones that have been established, Mb-scale deletions carried by the bladder cancer cells are mostly absent in the fusion cells, but copy number gains contributed by the cancer cells have stayed. Fusion cells exhibit increased populations of mitotic cells with 3-polar spindles, indicative of genomic instability. They grow faster in vitro and exhibit higher colony formation in anchorage-independent growth assay in soft agar than the parent UMUC-3 does. Fusion cells develop tumors, after 4 weeks of time lag, as efficiently as the parent UMUC-3 does in xenograft experiments. 264 genes are identified whose expression is specifically altered in the fusion cells. Many of them are interferon-stimulated genes (ISG), but are activated in a manner independent of interferon. Among them, we show that PD-L1 is induced in fusion cells, and its knockout decreases tumorigenesis in a xenograft model. PD-L1 is induced in a manner independent of STAT1 known to regulate PD-L1 expression, but is regulated by histone modification, and is likely to inhibit phagocytosis by PD1-expressing macrophages, thus protecting cancer cells from immunological attacks. The fusion cells overexpress multiple cytokines including CCL2 that cause tumor progression by converting infiltrating macrophages to tumor-associated-macrophage (TAM). The results present mechanisms of how cell fusion promotes tumorigenesis, revealing a novel link between cell fusion and PD-L1, and underscore the efficacy of cancer immunotherapy.

Cdc7 kinase is required for postnatal brain development.

The evolutionally conserved Cdc7 kinase plays crucial roles in initiation of DNA replication as well as in other chromosomal events. To examine the roles of Cdc7 in brain development, we have generated mice carrying Cdc7 knockout in neural stem cells by using Nestin-Cre. The Cdc7Fl/Fl NestinCre mice were born, but exhibited severe growth retardation and impaired postnatal brain development. These mice exhibited motor dysfunction within 9 days after birth and did not survive for more than 19 days. The cerebral cortical layer formation was impaired, although the cortical cell numbers were not altered in the mutant. In the cerebellum undergoing hypoplasia, granule cells (CGC) decreased in number in Cdc7Fl/F l NestinCre mice compared to the control at E15-18, suggesting that Cdc7 is required for DNA replication and cell proliferation of CGC at mid embryonic stage (before embryonic day 15). On the other hand, the Purkinje cell numbers were not altered but its layer formation was impaired in the mutant. These results indicate differential roles of Cdc7 in DNA replication/cell proliferation in brain. Furthermore, the defects of layer formation suggest a possibility that Cdc7 may play an additional role in cell migration during neural development.

Correction: Hsiao et al. Claspin-Dependent and -Independent Chk1 Activation by a Panel of Biological Stresses. Biomolecules 2023, 13, 125.

In the original publication [...].

Catalytic domain-dependent and -independent transcriptional activities of the tumour suppressor histone H3K27 demethylase UTX/KDM6A in specific cancer types.

The histone H3K27 demethylase, UTX/KDM6A, plays a critical role in the early development of vertebrates, and mutations are frequently found in various cancers. Several studies on developmental and cancer biology have focused on preferential transcriptional regulation by UTX independently of its H3K27 demethylase catalytic activity. Here, we analysed gene expression profiles of wild-type (WT) UTX and a catalytic activity-defective mutant in 786-O and HCT116 cells and confirmed that catalytic activity-dependent and -independent regulation contributes to the expression of most of the target genes. Indeed, the catalytic activity-defective mutant indeed suppressed colony formation similar to the WT in our assay system. However, the expression of several genes was significantly dependent on the catalytic activity of UTX in a cell type-specific manner, which could account for the inherent variation in the transcriptional landscape of various cancer types. The promoter/enhancer regions of the catalytic activity-dependent genes identified here were found to be preferentially modified with H3K4me1 and less with H3K27me3 than those of the independent genes. These findings, combined with previous reports, highlight not only the understanding of determinants for the catalytic activity dependency but also the development and application of pharmaceutical agents targeting the H3K27 or H3K4 modifications.

Water-Soluble Gd(III)-Porphyrin Complexes Capable of Both Photosensitization and Relaxation Enhancement.

With the aim of developing more stable Gd(III)-porphyrin complexes, two types of ligands 1 and 2 with carboxylic acid anchors were synthesized. Due to the N-substituted pyridyl cation attached to the porphyrin core, these porphyrin ligands were highly water-soluble and formed the corresponding Gd(III) chelates, Gd-1 and Gd-2. Gd-1 was sufficiently stable in neutral buffer, presumably due to the preferred conformation of the carboxylate-terminated anchors connected to nitrogen in the meta position of the pyridyl group helping to stabilize Gd(III) complexation by the porphyrin center. 1H NMRD (nuclear magnetic relaxation dispersion) measurements on Gd-1 revealed high longitudinal water proton relaxivity (r1 = 21.2 mM-1 s-1 at 60 MHz and 25 °C), which originates from slow rotational motion resulting from aggregation in aqueous solution. Under visible light irradiation, Gd-1 showed extensive photoinduced DNA cleavage in line with efficient photoinduced singlet oxygen generation. Cell-based assays revealed no significant dark cytotoxicity of Gd-1, while it showed sufficient photocytotoxicity on cancer cell lines under visible light irradiation. These results indicate the potential of this Gd(III)-porphyrin complex (Gd-1) as a core for the development of bifunctional systems acting as an efficient photodynamic therapy photosensitizer (PDT-PS) with magnetic resonance imaging (MRI) detection capabilities.

Aberrant association of chromatin with nuclear periphery induced by Rif1 leads to mitotic defect.

The architecture and nuclear location of chromosomes affect chromatin events. Rif1, a crucial regulator of replication timing, recognizes G-quadruplex and inhibits origin firing over the 50-100-kb segment in fission yeast, Schizosaccharomyces pombe, leading us to postulate that Rif1 may generate chromatin higher order structures inhibitory for initiation. However, the effects of Rif1 on chromatin localization in nuclei have not been known. We show here that Rif1 overexpression causes growth inhibition and eventually, cell death in fission yeast. Chromatin-binding activity of Rif1, but not recruitment of phosphatase PP1, is required for growth inhibition. Overexpression of a PP1-binding site mutant of Rif1 does not delay the S-phase, but still causes cell death, indicating that cell death is caused not by S-phase problems but by issues in other phases of the cell cycle, most likely the M-phase. Indeed, Rif1 overexpression generates cells with unequally segregated chromosomes. Rif1 overexpression relocates chromatin near nuclear periphery in a manner dependent on its chromatin-binding ability, and this correlates with growth inhibition. Thus, coordinated progression of S- and M-phases may require regulated Rif1-mediated chromatin association with the nuclear periphery.

Claspin-Dependent and -Independent Chk1 Activation by a Panel of Biological Stresses.

Replication stress has been suggested to be an ultimate trigger of carcinogenesis. Oncogenic signal, such as overexpression of CyclinE, has been shown to induce replication stress. Here, we show that various biological stresses, including heat, oxidative stress, osmotic stress, LPS, hypoxia, and arsenate induce activation of Chk1, a key effector kinase for replication checkpoint. Some of these stresses indeed reduce the fork rate, inhibiting DNA replication. Analyses of Chk1 activation in the cell population with Western analyses showed that Chk1 activation by these stresses is largely dependent on Claspin. On the other hand, single cell analyses with Fucci cells indicated that while Chk1 activation during S phase is dependent on Claspin, that in G1 is mostly independent of Claspin. We propose that various biological stresses activate Chk1 either directly by stalling DNA replication fork or by some other mechanism that does not involve replication inhibition. The former pathway predominantly occurs in S phase and depends on Claspin, while the latter pathway, which may occur throughout the cell cycle, is largely independent of Claspin. Our findings provide evidence for novel links between replication stress checkpoint and other biological stresses and point to the presence of replication-independent mechanisms of Chk1 activation in mammalian cells.

Claspin is Required for Growth Recovery from Serum Starvation through Regulating the PI3K-PDK1-mTOR Pathway in Mammalian Cells.

Claspin plays multiple important roles in regulation of DNA replication as a mediator for the cellular response to replication stress, an integral replication fork factor that facilitates replication fork progression and a factor that promotes initiation by recruiting Cdc7 kinase. Here, we report a novel role of Claspin in growth recovery from serum starvation, which requires the activation of PI3 kinase (PI3K)-PDK1-Akt-mTOR pathways. In the absence of Claspin, cells do not proceed into S phase and eventually die partially in a ROS- and p53-dependent manner. Claspin directly interacts with PI3K and mTOR, and is required for activation of PI3K-PDK1-mTOR and for that of mTOR downstream factors, p70S6K and 4EBP1, but not for p38 MAPK cascade during the recovery from serum starvation. PDK1 physically interacts with Claspin, notably with CKBD, in a manner dependent on phosphorylation of the latter protein, and is required for interaction of mTOR with Claspin. Thus, Claspin plays a novel role as a key regulator for nutrition-induced proliferation/survival signaling by activating the mTOR pathway. The results also suggest a possibility that Claspin may serve as a common mediator that receives signals from different PI3K-related kinases and transmit them to specific downstream kinases.

Histone Modification Analysis of Low-Mappability Regions.

Posttranslational modifications of histone are intimately related to chromatin/chromosome-mediated cellular events. Among all, the roles of histone modifications including acetylation, methylation, ubiquitination, and SUMOylation of lysine or arginine residue of nucleosome core histones in gene expression have been intensively studied. Genome-wide profiles of histone modification marks revealed their combinatorial organization in the functional features of chromatin. Analysis of histone modification by chromatin immunoprecipitation (ChIP) is one of the standard assays to examine chromatin states. Although high-throughput sequencing analysis (ChIP-seq) is now widely conducted, classical ChIP-qPCR analysis has advantages in investigation of multiple histone modification marks at a target site simply through the use of relatively small numbers of cells. Since ChIP-qPCR is devoid of biases caused by overamplification and inaccurate mapping of sequencing reads, it is a more reliable quantification method than genome-wide ChIP-seq especially for analyses of the low-mappability regions, which harbor many repetitive sequences and/or highly homologous segmental multiplications as found in gene clusters. We have recently analyzed histone H3 and H4 modifications of the Zscan4 family gene loci in an 880 kb gene cluster and found that the atypical enhancer-like structure is formed upon derepression of Zscan4. In this chapter, we describe the detailed protocols for histone modification ChIP-assay of repeat-enriched gene cluster regions. The protocol here we applied to mouse ES cells, but the protocol is perfectly applicable to human cultured cells and specimens.

Replicon hypothesis revisited.

Nearly 70 years after the proposal of semiconservative replication of generic material by Watson and Crick, we now understand many of the proteins involved in the replication of host chromosomes and how they operate. The initiator and replicator, proposed in the replicon hypothesis, are now well defined in both prokaryotes and eukaryotes. On the other hand, studies in prokaryotes and Archaea indicate alternative modes of initiation, which may not depend on an initiator. Here I summarize recent progress in the field of DNA replication and discuss the evolution of replication systems.

TT-pocket/HIRAN: binding to 3'-terminus of DNA for recognition and processing of stalled replication forks.

Stalled replication forks need to be swiftly detected and protected from collapse and the cause for fork stall be removed to restore the active replication fork. In bacteria, stalled forks are recognized and stabilized by PriA, a DEXH-type helicase, which also facilitates reassembly of an active replication fork. A TT-pocket (three-prime terminus binding pocket) present in the N-terminal segment of PriA plays a crucial role in stabilization of the stalled forks by specifically binding to the 3$^\prime$-terminus of the nascent leading strand. Eukaryotic proteins, Rad5/HLTF, contain a TT-pocket related domain, HIRAN, that specifically binds to 3'-terminus of DNA and play a role in stalled fork processing. While the TT-pocket of PriA facilitates the formation of an apparently stable and immobile complex on a fork with a 3'-terminus at the fork junction, HIRAN of Rad5/HLTF facilitates fork regression by itself. A recent report shows that HIRAN can displace 3 nucleotides at the end of the duplex DNA, providing mechanistic insight into how stalled forks are reversed in eukaryotes. In this article, I will compare the roles of 3'-terminus binding domains in stalled fork processing in prokaryotes and in eukaryotes.

Hypoxia-inducible factor-1α and poly [ADP ribose] polymerase 1 cooperatively regulate Notch3 expression under hypoxia via a noncanonical mechanism.

Upregulation of Notch3 expression has been reported in many cancers and is considered a marker for poor prognosis. Hypoxia is a driving factor of the Notch3 signaling pathway; however, the induction mechanism and role of hypoxia-inducible factor-1α (HIF-1α) in the Notch3 response are still unclear. In this study, we found that HIF-1α and poly [ADP-ribose] polymerase 1 (PARP-1) regulate Notch3 induction under hypoxia via a noncanonical mechanism. In the analyzed cancer cell lines, Notch3 expression was increased during hypoxia at both the mRNA and protein levels. HIF-1α knockdown and Notch3 promoter reporter analyses indicated that the induction of Notch3 by hypoxia requires HIF-1α and also another molecule that binds the Notch3 promoter's guanine-rich region, which lacks the canonical hypoxia response element. Therefore, using mass spectrometry analysis to identify the binding proteins of the Notch3 promoter, we found that PARP-1 specifically binds to the Notch3 promoter. Interestingly, analyses of the Notch3 promoter reporter and knockdown of PARP-1 revealed that PARP-1 plays an important role in Notch3 regulation. Furthermore, we demonstrate that PARP inhibitors, including an inhibitor specific for PARP-1, attenuated the induction of Notch3 by hypoxia. These results uncover a novel mechanism in which HIF-1α associates with PARP-1 on the Notch3 promoter in a hypoxia response element-independent manner, thereby inducing Notch3 expression during hypoxia. Further studies on this mechanism could facilitate a better understanding of the broader functions of HIF-1α, the roles of Notch3 in cancer formation, and the insights into novel therapeutic strategies.

Loss of full-length DNA replication regulator Rif1 in two-cell embryos is associated with zygotic transcriptional activation.

Rif1 regulates DNA replication timing and double-strand break repair, and its depletion induces transcriptional bursting of two-cell (2C) zygote-specific genes in mouse ES cells. However, how Rif1 regulates zygotic transcription is unclear. We show here that Rif1 depletion promotes the formation of a unique Zscan4 enhancer structure harboring both histone H3 lysine 27 acetylation (H3K27ac) and moderate levels of silencing chromatin mark H3K9me3. Curiously, another enhancer mark H3K4me1 is missing, whereas DNA methylation is still maintained in the structure, which spreads across gene bodies and neighboring regions within the Zscan4 gene cluster. We also found by function analyses of Rif1 domains in ES cells that ectopic expression of Rif1 lacking N-terminal domain results in upregulation of 2C transcripts. This appears to be caused by dominant negative inhibition of endogenous Rif1 protein localization at the nuclear periphery through formation of hetero-oligomers between the N-terminally truncated and endogenous forms. Strikingly, in murine 2C embryos, most of Rif1-derived polypeptides are expressed as truncated forms in soluble nuclear or cytosolic fraction and are likely nonfunctional. Toward the morula stage, the full-length form of Rif1 gradually increased. Our results suggest that the absence of the functional full-length Rif1 due to its instability or alternative splicing and potential inactivation of Rif1 through dominant inhibition by N-terminally truncated Rif1 polypeptides may be involved in 2C-specific transcription program.

Discovery of AS-0141, a Potent and Selective Inhibitor of CDC7 Kinase for the Treatment of Solid Cancers.

CDC7, a serine-threonine kinase, plays conserved and important roles in regulation of DNA replication and has been recognized as a potential anticancer target. We report here the optimization of a series of furanone analogues starting from compound 1 with a focus on ADME properties suitable for clinical development. By replacing the 2-chlorobenzene moiety in 1 with various aliphatic groups, we identified compound 24 as a potent CDC7 inhibitor with excellent kinase selectivity and favorable oral bioavailability in multiple species. Oral administration of 24 demonstrated robust in vivo antitumor efficacy in a colorectal cancer xenograft model. Compound 24 (AS-0141) is currently in phase I clinical trials for the treatment of solid cancers.

Extracellular DJ-1 induces sterile inflammation in the ischemic brain.

Inflammation is implicated in the onset and progression of various diseases, including cerebral pathologies. Here, we report that DJ-1, which plays a role within cells as an antioxidant protein, functions as a damage-associated molecular pattern (DAMP) and triggers inflammation if released from dead cells into the extracellular space. We first found that recombinant DJ-1 protein induces the production of various inflammatory cytokines in bone marrow-derived macrophages (BMMs) and dendritic cells (BMDCs). We further identified a unique peptide sequence in the αG and αH helices of DJ-1 that activates Toll-like receptor 2 (TLR2) and TLR4. In the ischemic brain, DJ-1 is released into the extracellular space from necrotic neurons within 24 h after stroke onset and makes direct contact with TLR2 and TLR4 in infiltrating myeloid cells. Although DJ-1 deficiency in a murine model of middle cerebral artery occlusion did not attenuate neuronal injury, the inflammatory cytokine expression in infiltrating immune cells was significantly decreased. Next, we found that the administration of an antibody to neutralize extracellular DJ-1 suppressed cerebral post-ischemic inflammation and attenuated ischemic neuronal damage. Our results demonstrate a previously unknown function of DJ-1 as a DAMP and suggest that extracellular DJ-1 could be a therapeutic target to prevent inflammation in tissue injuries and neurodegenerative diseases.

Regulation of HP1 protein by phosphorylation during transcriptional repression and cell cycle.

HP1 (heterochromatin protein 1), a key factor for the formation of heterochromatin, binds to the methylated lysine 9 of histone H3 (H3K9me) and represses transcription. While the H3K9me mark and HP1 binding are thought to be faithfully propagated to daughter cells, the heterochromatin structure could be dynamically regulated during cell cycle. As evidenced by the well-known phenomenon called position effect variegation (PEV), heterochromatin structure is dynamically and stochastically altered during developmental processes, and thus the expression of genes within or in the vicinity of heterochromatin could be affected by mutations in factors regulating DNA replication as well as by other epigenetic factors. Recent reports show that HP1 also plays an important role in the maintenance and transmission of chromosomes. Like many other factors ensuring faithful chromosome segregation, HP1 family proteins are subjected to posttranslational modifications, most notably phosphorylation, in a cell cycle-dependent manner. Recent studies identified a conserved phosphorylation site that profoundly affects the functions of HP1 during mitotic phase. In this commentary, we discuss dynamic regulation of HP1 protein by phosphorylation during transcriptional repression and cell cycle.

A KDM6 inhibitor potently induces ATF4 and its target gene expression through HRI activation and by UTX inhibition.

UTX/KDM6A encodes a major histone H3 lysine 27 (H3K27) demethylase, and is frequently mutated in various types of human cancers. Although UTX appears to play a crucial role in oncogenesis, the mechanisms involved are still largely unknown. Here we show that a specific pharmacological inhibitor of H3K27 demethylases, GSK-J4, induces the expression of transcription activating factor 4 (ATF4) protein as well as the ATF4 target genes (e.g. PCK2, CHOP, REDD1, CHAC1 and TRIB3). ATF4 induction by GSK-J4 was due to neither transcriptional nor post-translational regulation. In support of this view, the ATF4 induction was almost exclusively dependent on the heme-regulated eIF2α kinase (HRI) in mouse embryonic fibroblasts (MEFs). Gene expression profiles with UTX disruption by CRISPR-Cas9 editing and the following stable re-expression of UTX showed that UTX specifically suppresses the expression of the ATF4 target genes, suggesting that UTX inhibition is at least partially responsible for the ATF4 induction. Apoptosis induction by GSK-J4 was partially and cell-type specifically correlated with the activation of ATF4-CHOP. These findings highlight that the anti-cancer drug candidate GSK-J4 strongly induces ATF4 and its target genes via HRI activation and raise a possibility that UTX might modulate cancer formation by regulating the HRI-ATF4 axis.

G-quadruplex binding protein Rif1, a key regulator of replication timing.

DNA replication is spatially and temporally regulated during S phase to execute efficient and coordinated duplication of entire genome. Various epigenomic mechanisms operate to regulate the timing and locations of replication. Among them, Rif1 plays a major role to shape the 'replication domains' that dictate which segments of the genome are replicated when and where in the nuclei. Rif1 achieves this task by generating higher-order chromatin architecture near nuclear membrane and by recruiting a protein phosphatase. Rif1 is a G4 binding protein, and G4 binding activity of Rif1 is essential for replication timing regulation in fission yeast. In this article, we first summarize strategies by which cells regulate their replication timing and then describe how Rif1 and its interaction with G4 contribute to regulation of chromatin architecture and replication timing.